Galectin-8 and galectin-8-like proteins and DNA molecules coding therefor

ABSTRACT

The invention relates to a new mammalian S-type lectin, termed galectin-8, and to galectin-8-like proteins, to fragments thereof, to DNA molecules coding therefor and to pharmaceutical compositions comprising said proteins. Galectin-8, a widely expressed protein of 35 kDa is shown to be implicated in regulation of cell growth, particularly in inhibition of cell proliferation.

FIELD OF THE INVENTION

The present invention is generally in the field of mammalian S-typelectin proteins, now designated galectins, which are thiol-dependent andspecifically bind β-galactoside residues.

More specifically, the present invention relates to a new S-typemammalian lectin, termed hereinafter as "galectin-8", and togalectin-8-like proteins, to DNA molecules coding therefor and toantibodies raised against said proteins. The invention further relatesto pharmaceutical compositions comprising said proteins for the purposeof cell growth regulation in general, and more particularly forinhibition of cell proliferation and for treatment of tumors.

BACKGROUND OF THE INVENTION

Lectins are involved in a wide variety of cellular functions, many ofwhich are related to their only common feature, the ability to bindcarbohydrates specifically and reversibly, and to agglutinate cellsreviewed in (1)!. Animal lectins are classified as C-lectins, which areCa²⁺ -dependent and are structurally related to the asialoglycoproteinreceptor, and galectins, previously known as S-type lectins, which arethiol-dependent and specifically bind β-galactoside residues. Inmammals, four galectin types have been sequenced and characterized, andthere is evidence for the existence of other relatives (2,3). All knownmembers of this family lack a signal peptide, are found in the cytosol,and are isolated as soluble proteins. However, there is evidence thatsome members are externalized by an atypical secretory mechanism.

Galectins require fulfillment of two criteria: affinity forβ-galactosides and significant sequence similarity in the carbohydraterecognition domain (CRD) (4), the relevant amino acids residues of whichhave been determined by X-ray crystallography (5). Galectin-1 and -2 arehomodimers with subunit molecular weight of ≈14 kDa, that are notsubjected to post-translational modifications (6). Galectin-1 is foundin the extracellular matrix and has been shown to interact with laminin(7). The function of galectin-1 and -2 is not yet fully understood,although there is evidence that they might be involved in regulation ofcell growth (8); cell adhesion (7); cell transformation (9); andembryogenesis (10).

Larger galectins (galectin-3) (previously known as CBP-35, Mac-2, RL-29)do exist ((11) and references therein). These are monomeric 29-35 kDamosaic proteins, composed of an N-terminal half made of tandem repeatscharacteristic of the collagen gene superfamily, and a C-terminal halfhomologous to galectins-1 and -2 (11). Galectin-3 also binds laminin,and is implicated as component of growth regulatory systems; mediator ofcell--cell and cell-matrix interactions; modulator of immune response;marker of neoplastic transformation, and indicator for metastaticpotential of melanoma cells.

Galectin-4 was cloned from rat intestine (12), and an homologous proteinwas cloned from nematode (13). Galectin-4 is a monomer with molecularmass of 36 kDa. It contains tandem domains of ≈140 amino-acids each,homologous to galectin-1 and -2, that are separated by a link region(12). The function of galectin-4 is presently unknown.

Galectins may functionally substitute each other. The absence of anymajor phenotypic abnormalities in mice carrying a null mutation in thegene encoding galectin-1, suggests that other protein(s), presumablygalectin-3, are capable of functionally substituting for galectin-1, atleast at early stages of embryogenesis.

It is an object of the present invention to provide the cloning of acDNA encoding for a novel protein that we term galectin-8. Galectin-8has the characteristic properties of other galectins (2,3), and it isstructurally related (34% identity) to rat galectin-4 (12).

SUMMARY OF THE INVENTION

According to the present invention, a novel protein of 35 Kd which hasthe characteristic properties of galectins (S-type mammalian lectins)was cloned from a rat liver cDNA expression library. This protein wasoriginally called by us RL-30 protein. However, the nomenclature ofS-type lectins has recently been changed to galectins (2). Since namesfor galectins 1-7 were already assigned (3), this new protein has nowbeen named galectin-8, but it is to be understood that this is the sameprotein formerly called by us RL-30.

Thus, in one embodiment, the present invention provides a biologicallyactive S-type lectin named galectin-8 and galectin-8-like proteins andfragments thereof selected from:

(i) the protein galectin-8 of the amino acid sequence depicted in FIG. 1(SEQ ID NO:2);

(ii) a protein having greater than about 80 percent similarity to all orpart of the sequence of amino acid residues 1-316 depicted in FIG. 1(residues 1-316 of SEQ ID NO:2);

(iii) a protein having greater than about 80 percent similarity to allor part of the sequence of amino acid residues 1-151 depicted in FIG. 1(residues 1-151 of SEQ ID NO:2);

(iv) a protein having greater than about 80 percent similarity to all orpart of the sequence of amino acid residues 152-316 depicted in FIG. 1(residues 152-316 of SEQ ID NO:2);

(v) a protein of (i), (ii), (iii) or (iv) in which one or more aminoacid residues have been added, deleted, replaced or chemically modifiedwithout substantially affecting the biological activity of the protein;

(vi) a biologically active fragment of (i) to (v); and

(vii) an homologous polypeptide to that of (i) to (vi) derived fromanother mammal and which has a similar biological activity to that of(i) to (vi).

In another embodiment, the present invention relates to an isolated DNAsequence encoding galectin-8 or a galectin-8-like protein.

By one embodiment, the isolated DNA sequence of the invention is onethat encodes a polypeptide product of prokaryotic or eukaryotic hostexpression, said product having all or part of the primary structuralconformation of galectin-8 or of a galectin-8-like protein and havingthe biological activity of galectin-8.

The above DNA sequence of the invention may be any one of the groupconsisting of:

(i) a DNA molecule having a nucleotide sequence derived from the codingregion of a native galectin-8 or galectin-8-like gene;

(ii) a DNA molecule capable of hybridization to the cDNA clones of (i)under moderately stringent conditions and which encodes biologicallyactive galectin-8 or a galectin-8-like protein; and

(iii) a DNA molecule which differs, as a result of the degenerativenature of the genetic code, from the DNA sequences defined in (i) or(ii) and which encodes biologically active galectin-8 or agalectin-8-like protein.

By way of other embodiments, the above DNA sequence of the invention isone selected from:

(i) a DNA molecule comprising the coding nucleic acid sequence depictedin FIG. 1 (nucleotides 121-1068 of SEQ ID NO:1);

(ii) a DNA molecule having the nucleic acid sequence of (i) in which oneor more codons has been added, replaced or deleted in a manner that thepolypeptide encoded by said sequence essentially retains the samebiological properties as the polypeptide encoded by an unaltered DNAsequence;

(iii) a DNA molecule encoding a polypeptide having an amino acidsequence of a polypeptide encoded by the DNA molecule of (i) or (ii) butwhich differs therefrom in view of the degenerative nature of thegenetic code;

(iv) a DNA molecule having a coding nucleotide sequence, which ishomologous to the DNA molecule of (i), (ii) or (iii), which is derivedfrom a mammal other than rats and which encodes a polypeptide having asimilar biological activity to that encoded by the sequences of (i),(ii) or (iii);

(v) a fragment of the coding sequence of (i)-(iv) which encodes apolypeptide which essentially retains the biological properties of thepolypeptide encoded by the unfragmented DNA molecule; and

(vi) a DNA molecule comprising the coding DNA sequence of a fragment of(i)-(v) and additional DNA sequences in the 3' and 5' ends.

In a further embodiment, the present invention relates to a recombinantDNA molecule comprising a coding sequence according to any of (i)-(iii)and (i)-(iv) above or a fragment thereof according to (v) or (vi) above.

The present invention also provides a recombinant expression vectorcomprising any one of the above-mentioned DNA molecules of theinvention. Such a recombinant expression vector may be one capable ofbeing expressed in prokaryotic or eukaryotic hosts, the vectorcontaining, in addition to any one of the above galectin-8 orgalectin-8-like protein encoding sequences, various other sequences suchas, for example, those sequences that are known to be important forexpression of the desired sequence and the maintenance and propagationof the vector in the host cell. Construction of such recombinantexpression vectors is by way of any of the known procedures.

The present invention further provides a method for preparing galectin-8or a galectin-8-like protein or a biologically active fragment thereof,comprising culturing a suitable host cell containing the aboverecombinant vector of the invention under conditions promotingexpression.

The protein of the invention may be prepared, as noted above, byexpression of a recombinant vector comprising a DNA sequence encodingthe protein, or it may be isolated and purified from various mammaliantissues using standard procedures for protein extraction andpurification. In such purification procedures there may be employed yetanother aspect of the present invention, namely, antibodies which areimmunoreactive with native or recombinant galectin-8 or with agalectin-8-like protein. Such antibodies may be applied in standardaffinity chromatography methods to provide for the final purificationsteps of the galectin from various tissues. The preparation of theantibodies is by standard procedures using native or recombinantgalectin-8 or a fragment thereof or a galectin-8-like protein or afragment thereof as antigen or immunogen to stimulate antibodyproduction in suitable animals. Both polyclonal and monoclonalantibodies to galectin-8 are encompassed by the invention. Theseantibodies can be prepared by standard procedures well-known in the art.

The anti-galectin-8 antibodies of the invention may also be employed inan assay method for the detection of overexpression of galectin-8 inmammalian tissue, said method comprising applying an effective amount ofthe antibodies to a tissue or body fluid sample obtained from a mammaland determining the extent of antibody binding to the sample. In such anassay, standard procedures may be employed, such as, for example, ELISAassay procedures.

In addition, the present invention also provides pharmaceuticalcompositions comprising as active ingredient an effective amount ofgalectin-8 or of a mammalian galectin-8-like protein and a suitablediluent or carrier, in particular compositions for cell growthregulation, more specifically for the inhibition of cell proliferation,for example for the treatment of cancer.

In these above compositions the diluents or carriers may be any of thosesubstances well known in the art for the preparation of pharmaceuticalcompositions, and likewise the compositions may be prepared by standardprocedures. Actual dosages and modes of administration of the abovecompositions are to be determined by skilled professionals.

DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the cDNA sequence (SEQ ID NO:1) of galectin-8 and deducedprotein sequence (SEQ ID NO:2). The cDNA sequence of 1247 base pairs(bp) contains an open reading frame from 121-1068 bp, which encodes fora protein of 316 amino acids.

FIGS. 2A-2B show that galectin-8 encodes for a galectin with twohomologous carbohydrate-binding regions. A schematic structure ofgalectin-8 is presented (top). Each box represents a putativecarbohydrate-binding domain, linked by a 32 amino acid long peptide.Shown are invariant amino acids preserved in most galectins analyzed sofar, e.g. SEQ ID NO:19 in the boxed N-terminal carbohydrate-bindingdomain. The Arg residue, indispensable for sugar binding, located at theC-terminal carbohydrate recognition domain (CRD), and its correspondingIle residue, localized to the N-terminal CRD, are underlined. Amino acidsequences of different galectins are presented for comparison (bottom).These include: human galectin-1 (Galec-1) (SEQ ID NO:3); humangalectin-2 (Galec-2) (SEQ ID NO:4); the carbohydrate binding domain(amino acids 128-263) of rat galectin-3 (Galec-3) (SEQ ID NO:5);N-terminal (Galec-4-Nt) (SEQ ID NO:6) and C-terminal (Galec-4-Ct) (SEQID NO:7) halves of galectin-4; N-terminal (CE-Nt) (SEQ ID NO:8) andC-terminal (CE-Ct) (SEQ ID NO:9) halves of a 32-kDaβ-galactoside-binding protein from Caenorhabditis elegans; N-terminal(Galec-8-Nt) (SEQ ID NO:10) and C-terminal (Galec-8-Ct) (SEQ ID NO:11)halves of galectin-8. Residues with shared identity are boxed. Residueswith shared similarity are shaded.

FIG. 3 shows Northern blot analysis of RNA from rat tissues probed withgalectin-8 cDNA. Top, 30 μg of total RNA from the indicated tissues waselectrophoresed, blotted, and probed with labeled galectin-8 PCR productas described in "Experimental Procedures". The migration of the 18S and28S rRNA are marked. Bottom, the same blot was stripped and reblottedwith cDNA encoding for GAPDH.

FIG. 4 shows immunoprecipitation of in-vitro translation product ofgalectin-8 by lp-lec8 antibodies. Fifty μl of the ³⁵ S-labeledgalectin-8, expressed as in-vitro translation product (see "ExperimentalProcedures"), were immunoprecipitated by lp-lec8 antibodies as describedin Example 2 herein. Five μl of the total ³⁵ S-labeled galectin-8(total), 5 μl of the fraction not precipitated by the antibodies (Sup),and 50 μl of the immunoprecipitated fraction (IP) were subjected to 12%SDS-PAGE and autoradiography.

FIG. 5 shows binding of tag-free recombinant galectin-8 (r-galectin-8)to lactosyl-Sepharose. Tag-free r-galectin-8 was expressed in pLysS asdescribed under "Experimental Procedures". After centrifugation, 30 mlof the soluble bacterial proteins were purified over 5 ml oflactosyl-Sepharose. r-galectin-8 was eluted with 100 mM lactose inbuffer-I, and 1 ml fractions were collected. Ten μl of the total andeffluent fractions and 50 μl from each elution fraction were resolved by12% SDS-PAGE, transferred to nitrocellulose and Western immunoblottedwith lp-lec8 antibodies.

FIGS. 6A and 6B show binding of rat hepatic galectin-8 tolactosyl-Sepharose. Five g of rat liver were homogenized in buffer-I asdescribed under "Experimental Procedures" and cytosolic extracts (25 ml)were applied over 5 ml of lactosyl-Sepharose. After extensive washingthe bound proteins were eluted with 100 mM lactose in buffer-I. One mlfractions (numbered 1-10) were collected and frozen for a period of 16 hat -20° C. Eluted fractions (N° 3-5) were toughed, centrifuged for 15min at 12000×g and the pellets were resuspended in 50 ml sample buffer(34) . Ten μg protein of total (A) and effluent (B) fractions as well as50 μl of the supernatant (C) and resuspended pellet (D) of the elutedfractions (N° 3-5) were resolved by 12% SDS-PAGE, transferred tonitrocellulose and Western immunoblotted with lp-lec8 antibodies (FIG.6A), or subjected to Coomasie staining (FIG. 6B).

FIG. 7 shows inhibition by recombinant galectin-8 of serum-induced ³H-thymidine incorporation into DNA. ³ H-Thymidine incorporation into DNAwas examined as follows: Confluent CHO cells, grown in 24-well trays(Costar), were starved for 48 hours in serum-free medium. Mediumcontaining 10% fetal calf serum was added to the cells in the presenceof rgalectin-8 at the indicated concentrations, and the cells wereincubated for 14.5 hours at 37° C. The medium was then washed, and thecells were incubated for 2 hours at 37° C. in 1 ml of serum-free mediumcontaining 1% BSA, 20 mM Hepes (pH 7.5) and 0.5 μCi/ml ³ H!-thymidine.At the end of incubation, the solution was removed, cells were washed 3times in ice-cold PBS and incubated for 30 min at 4° C. in 0.5 mlice-cold 7.5% trichloroacetic acid. The pellets were washed twice with98% ice-cold ethanol, dissolved in 0.6 ml of 0.1 M NaOH, and countedusing scintillation cocktail containing xylene and Lumax (6:4 ratio,respectively).

FIG. 8 shows chromatography of galectin-8 over a FPLC column.Approximately 100 μg protein was loaded onto Superdex-200 HR (Pharmacia)FPLC column equilibrated with buffer A (PBS, 4 mM β-mercaptoethanol, 2mM EDTA), and run for 60 min at 0.5 ml/min. O.D. was measured at 215 nm,and the column profile was obtained by running separately standardmarks.

DETAILED DESCRIPTION OF THE INVENTION

Galectin-8 is a novel, widely expressed protein of 35 kDa which has thecharacteristic properties of galectins (S-type mammalian lectins). Threelines of evidence demonstrate that galectin-8 is indeed a novelgalectin: i. its deduced amino acid sequence contains two domains withconserved motifs that are implicated in the carbohydrate binding ofgalectins; ii. in vitro translation products of galectin-8 cDNA orbacterially-expressed recombinant galectin-8 are biologically active andpossess sugar-binding and hemagglutination activity; iii. a protein ofthe expected size (34 kDa), that binds to lactosyl-Sepharose and reactswith galectin-8-specific antibodies is present in rat liver andcomprises 0.025% of the total Triton-soluble hepatic proteins.

Overall, galectin-8 is structurally related (34% identity) togalectin-4, a soluble rat galectin with two carbohydrate-binding domainsin the same polypeptide chain, joined by a link peptide. Nonetheless,several important features distinguish these two galectins: i. Northernblot analysis revealed that unlike galectin-4 that is confined to theintestine and stomach, galectin-8 is expressed in liver, kidney, cardiacmuscle, lung, and brain; ii. unlike galectin-4, but similar togalectins-1 and -2, galectin-8 contains 4 Cys residues; iii. the linkpeptide of galectin-8 is unique and bears no similarity to any knownprotein; iv. the N-terminal carbohydrate-binding region (CRD) ofgalectin-8 contains a unique WG-E-I motif instead of the consensusWG-E-R/K motif implicated as playing an essential role in sugar-bindingof all galectins. Together with galectin-4, galectin-8 thereforerepresents a subfamily of galectins consisting of a tandem repeat ofstructurally different CRDs within a single polypeptide chain.

As used herein, the term "galectin-8-like protein" refers to a proteinderived from any mammal, including humans, which protein presentshomology to galectin-8 as defined in the present invention and has thebiological properties of galectin-8.

Galectin-8 was cloned when a λ-ZAP rat liver cDNA library was screenedwith affinity-purified antibodies directed against a 14-amino acidpeptide located at the C-terminal end of the insulin-receptor substrate1 (IRS-1) (14). Since galectin-8 bears no sequence similarity either toIRS-1, or to the peptide used as immunogen, it was suspected that thereactivity towards IRS-1 antibodies could be due to a false positivereaction. This conclusion is supported by the fact that the anti-peptideantibodies used for screening, failed to react with purified recombinantgalectin-8 either by means of immunoprecipitation, or immunoblotting.

The primary structure of galectin-8 resembles that of galectin-4,namely, two homologous (38% identity) carbohydrate-binding regions(CRDs) linked by a short ≈30-amino acids linking peptide. This uniquearchitecture is shared so far only by two galectins: rat galectin-4 (12)and its C. elegans homologue (13). Other galectin types, that contain asingle CRD, exist and function as non-covalent dimers, which providesthem with the potential to aggregate or agglutinate glycoconjugates.Since galectin-4 exists as a monomer, experiments were carried out todetermine whether galectin-8 exists as a monomer or a dimer. Separationof galectin-8 over Superdex-200 HR (Pharmacia) FPLC column according tothe present invention revealed that galectin-8 exists as a monomer (FIG.8). Hepatic galectin-8 (FIG. 6) has a similar mobility on SDS-PAGE asits recombinant counterpart (FIG. 5). This suggests, though not proves,that hepatic galectin-8 is neither heavily glycosylated, nor it issubjected to extensive post-translational modifications (e. g.phosphorylation).

Although galectin-8 contains two putative CRDs, potential differences insugar-binding between the domains is predicted from a criticaldifference in their sequence WG-E-I vs. WG-E-R at the N- and C-terminalCRDs of galectin-8, respectively (cf. FIG. 2)!. The (underlined) Argresidue has been implicated as playing an important role in theinteractions between galectins and the glucose moiety of lactose (5).Furthermore, site-directed mutagenesis studies (4) indicate that thisconserved Arg is indispensable for sugar binding. The presence of Ile⁹⁰(instead of an Arg) at the N-terminal CRD of galectin-8 suggests thatthis domain might have a different sugar-binding specificity. In thatrespect galectin-8 resembles galectin-4 whose CRDs are distinct both instructure and sugar-binding specificity (12). The presence of two CRDswith a potentially different sugar-binding specificity might be requiredto achieve high affinity binding to multivalent glycoprotein ligandspossessing different sugar moieties.

Like other galectins, galectin-8 lacks a classical signal sequence or atransmembrane segment. Indeed, galectin-8 was isolated from thecytosolic fraction of rat liver. These findings do not exclude thepossibility that galectin-8, like other galectins, could be externalizedby an atypical secretory mechanism (15). Immunohistochemical studiesrevealed that secreted galectins are concentrated in evaginations of theplasma membrane, which pinch off to form labile lectin-richextracellular vesicles which may interact with cell surface proteins(15). Expression of galectin-8 seems to be developmentally regulated.Very low levels of expression were noted in whole embryos, while highlevels of expression were noted in adult tissues. In that respectgalectin-8 might resemble other galectins that were implicated asregulators of cell growth and embryogenesis (8-10).

The invention will now be described by way of the following non-limitingexamples and the accompanying drawings.

EXAMPLES Experimental Procedures

(a) Materials--Restriction enzymes were purchased from Fermentas.Radiolabeled nucleotides and ³⁵ S!methionine were from Amersham(Amersham, Buckinghamshire, UK). All other reagents were from Sigmaunless stated otherwise.

(b) Antibodies--Antisera to insulin receptor substrate 1 (anti-IRS-1)were raised in rabbits according to standard procedures, by injection ofa peptide Cys-Tyr-Ala-Ser-Ile-Asn-Phe-Gln-Lys-Gln-Pro-Glu-Asp-Arg-Gln(SEQ ID NO:12) corresponding to the carboxy-terminal 14 amino acids ofrat liver IRS-1 (and an additional Cys residue at the N-terminal site).Antibodies were affinity-purified from the serum by adsorption onto acolumn of peptide coupled to Affi-gel 10, elution with 100 mM HClglycine pH 2.7, and immediate neutralization. Antiglutathione-S-transferase (GST) antisera was a kind gift from Y. Yarden(Weizmann Institute).

(c) Screening of Rat Liver cDNA Expression Library--λ-Zap rat liver cDNAlibrary in the Lambda ZAP II Vector (Stratagene, La Jolla, Calif.), wasscreened separately and in duplicate with affinity-purified anti IRS-1antibodies (see (b) above). Screening was carried out according to theinstruction manual provided by the manufacturer (picoBlue™Immunoscreening Kit, Stratagene, La Jolla, Calif.). Positive plaqueswere isolated by three repetitive cycles of the procedure. TheExAssist/SOLR system (Stratagene, La Jolla, Calif.) was used to allowefficient excision of the Bluescript phagemid from the λ-ZAP vector, andSOLR cells containing positive clones were isolated. Initial DNAsequencing of one positive clone was carried on both strands, using T3and T7 universal primers with Sequenase version 2.0, (United StatesBiochemicals, Cleveland, Ohio). Subsequent sequencing was carried outwith internal primers designed as the sequencing progressed. All othermanipulations of nucleic acids such as restriction, ligations,transformation, gel electrophoresis, blotting, gel elution,radiolabeling, and preparation of buffers were done using standardprotocols (16). Search of the GenBank revealed that the isolated cloneis unique and it bears no sequence similarity with IRS-1, or thepeptide, against which the antibodies were raised. The reason why thisclone was picked up by the antibodies remains unclear.

(d) Northern Blot Analysis--RNA extraction was carried out as described(16). Total RNA (30 μg) was electrophoresed, the gel was blotted ontonitrocellulose, and the blot was probed with labeled PCR product whichwas obtained by the following procedure. Two primers,5'-CCCGACAATCCCCTATGTCAGTACC-3 (SEQ ID NO:13) and5'-GCATGGCCAGGCCTGACAACA-3' (SEQ ID NO:14), were used to amplify theentire cDNA coding sequence of galectin-8, using the cloned cDNA inBluescript as a template. The PCR products were labeled with α-³² P!-ATPby random priming with DECAprime II DNA labeling kit (Ambion, Austin,Tex.). Hybridization was carried out at 42° C. in 50% formamide 5×SSC,and washes were at 60° C. in 0.1×SSC, 0.1% SDS.

(e) Expression of recombinant galectin-8 in Escherichia Coli--Expressionof galectin-8 as a GST fusion protein (GST-galectin-8) was carried outby using two primers: T7 and 5'-GGGGGGGGATCCATGTTGTCCTTAAGCAAT-3' (SEQID NO:15) (the EcoR I, Nde I, and BamH I restriction sites,respectively, in the primer are underlined) to amplify the entire cDNAinsert of galectin-8, using the cloned cDNA in Bluescript as a template.The PCR products were digested with BamH I and EcoR I, gel-purified, andligated into pGeX-2X expression plasmid (Pharmacia) in the TOP₁₀bacterial host (Invitrogen). For direct expression of (tag-free)r-galectin-8, a sense primer 5'-GGGGGGCATATGTTGTCCTTAAGCAAT-3' (SEQ IDNO:16) and an antisense primer 5'-GGGGGGGGATCCGCCATTTTGTATTTCCAG-3' (SEQID NO:17) were used to amplify the entire coding sequence of galectin-8,using the cloned cDNA in Bluescript as a template. The PCR products weredigested by Nde I and BamH I, gel-purified, and ligated into a pET-3aexpression plasmid (Novagen) in the pLysS bacterial host. Sequencing ofboth expression plasmids was carried out to ensure proper, in-frame,ligation of the inserts.

To express GST-galectin-8, bacteria were cultured in 0.5 liter of LBmedium until the absorbance at 600 nm was 0.5. Expression ofGST-galectin-8 was then induced with 5 mMisopropyl-1-thio-β-D-galactopyranoside (IPTG) for 4 h. To isolate therecombinant protein, a bacterial pellet was isolated by centrifugation,resuspended in 30 ml of buffer I (phosphate buffered saline containing 4mM β-mercaptoethanol, 2 mM EDTA, 10 μg/ml soybean trypsin inhibitor, 2mM benzamidine and 1 mM phenylmethylsulfonyl fluoride, pH 7.5), andlysed by sonication. Debris were removed by centrifugation at 38,000×gat 4° C. for 45 min., and 30 ml of the soluble extract were passed over5 ml of lactosyl-Sepharose. Unbound proteins were eluted with buffer I,while the lectin was subsequently eluted with buffer I containing 100 mMlactose. A similar procedure was utilized to express r-galectin-8 in thepET-3a expression plasmid, save for the fact that the bacteria werecentrifuged when the absorbance at 600 nm was 0.3, without addition ofIPTG. Recombinant galectin-8 was isolated under reducing conditions,since in their absence the protein underwent denaturation even whenmaintained at 4° C.

(f) In-Vitro translation of galectin-8--For in vitro translation ofgalectin-8, the BamH I/EcoR I-digested PCR product, described above, wascloned into pcDNA I mammalian expression plasmid (Invitrogen). In vitrotranslation in the presence of ³⁵ S!-methionine was performed using theTNT in vitro translation kit (Promega) according to the manufacturer'sinstructions.

(g) Immunoprecipitation--lp-lec8 antibodies were added to 60 μl of 50%protein A-Sepharose in 0.1 M Tris buffer, pH 8.5, and were incubated for1 hr at 4° C. Bacterial cell extracts were prepared in buffer I. 500 μlextracts (0.8 mg) were incubated for 2 hr with the antibody-proteinA-Sepharose complex. Immunocomplexes were washed, suspended in samplebuffer, resolved on 10-12% SDS-PAGE and transferred to nitrocellulosefor Western blotting.

(h) Protein electrophoresis and blotting--Immunoblotting was carried outby standard procedures. The blotted proteins were incubated with lp-lec8antibodies at 4° C. for 16 h and then were extensively washed. To detectantibody binding, a horseradish peroxidase-conjugated Protein A ECL kit(Amersham) was used according to the manufacturer's instructions.

(i) Purification of galectin-8 from rat liver--Freshly isolated ratlivers from male Wistar rats were homogenized in buffer I (1 g/5 ml)supplemented with 10 μg/ml aprotinin and 5 μg/ml leupeptin. Thehomogenate was centrifuged for 1 h at 4° C. at 100,000×g, and 25 ml ofthe supernatant were passed over 5 ml of lactosyl-Sepharose, followingthe procedure described above. The eluted fractions were kept frozen at-20° C. Since intact galectin-8 denatures upon freezing, the frozenfractions were toughed, and centrifuged at 12, 000×g, for 15 min. at 4°C. to precipitate, and thus concentrate, galectin-8. Supernatants andpellets were resuspended in sample buffer, resolved by 12% SDS-PAGE,transferred to nitrocellulose and Western immunoblotted with lp-lec8antibodies. The amount of galectin-8 in rat liver was estimated using100,000×g supernatants that were prepared in buffer I in the presence of1% Triton-X-100.

(j) Assay of lectin activity--The biological activity of galectin-8 wasassayed by measuring its ability to agglutinate formaldehyde-fixed,trypsin-treated rabbit erythrocyte. Rabbit erythrocytes weretrypsin-treated according to Lis and Sharon (17). Cells were incubatedfor 1 h at 37° C. with 0.1% trypsin in PBS, washed five times in 10volumes of 0.9% NaCl/packed ml of cells, and resuspended in 0.9% NaCl toyield an erythrocyte suspension with an absorbance of 1.5 at 620 nm.Half ml aliquots of erythrocyte suspension was incubated for 45 in atroom temperature with the lectin solution. Aliquots (0.2 ml) of theupper part of the tube were removed, mixed with 0.8 ml of PBS, and theoptical density at 620 nm was monitored.

EXAMPLE 1

Isolation of galectin-8, a novel mammalian galectin

A cDNA encoding for a new galectin, termed galectin-8, was cloned from aλ2-Zap rat liver cDNA library (FIG. 1). The isolated clone contained anopen reading frame (ORF) (nucleotides 112-1068) with a potentialinitiation ATG codon at position 121. This ORF coded for 316 aminoacids, which form a protein of about 35 kDa. The putative codingsequence was followed by a signal for translation termination (TAG) and176 nucleotides of 3'-untranslated region. Search of the GenBank forsimilar nucleotide sequences revealed that this sequence is unique. Thissequence, depicted in FIG. 1, has been submitted to the Gen Bank™/EMBLData Bank with accession number U09824.

Analysis of galectin-8 using alignment algorithms suggested the presenceof two homologous domains ≈140 amino acids each, linked by a linkpeptide of 32 amino acid residues (FIG. 2, top). Thirty eight percent ofthe amino acids were identical between the first and second domains(FIG. 2, bottom). Both domains contained sequence motifs (e. g. H-NPR;WG-EE) that have been conserved among most carbohydrate recognitiondomains (CRDS) of galectins analyzed so far. Structurally, galectin-8resembles a 32-kDa β-galactoside-binding protein from Caenorhabditiselegans (13) (CE-galectin), and rat galectin-4 (galectin-4) (12), thatalso contain two CRDs connected by a link peptide (FIG. 2). At the levelof nucleic acids, galectin-8 is 50% and 45% homologous to galectin-4 andCE-galectin, respectively. At the level of amino acids, galectin-8shares 34% and 31% identity, respectively, with the above proteins. Nohomology with any known protein was found in the region of the linkpeptide. Like other galectins, galectin-8 lacks classical signalsequence or transmembrane segment, but it contains three potentialN-linked glycosylation (Asn-X-Ser/Thr) sites. Analysis of its predictedsecondary structure (not shown), revealed that the N-and C-terminaldomains of galectin-8 share a great degree of structural homology, asexpected from their primary structure. Both domains are predicted toform several β-sheets, a structural feature of other galectins (5).

The cDNA clone encoding galectin-8 may be used as a probe to isolate andcharacterize the full length genomic sequence encoding this protein invarious mammals, for example, humans and rats, using standardprocedures.

Further, the above mentioned cDNA clone and/or the full-length genomicsequence encoding galectin-8 may be used to generate, by standardprocedures, fragments containing only a portion of the full-lengthgalectin-8 sequence, where each fragment essentially retains at leastone of the biological activities of galectin-8. These fragments aretermed `biologically active fragments`. Moreover the galectin-8 sequencemay also be used to generate analogs of galectin-8 (herein termed"galectin-8-like proteins") or fragments thereof, such analogs having atleast one amino acid residue added, deleted or replaced by another incomparison to the native galectin-8 sequence, and such analogsessentially retaining the biological activity of their non-modifiedprogenitor molecules.

EXAMPLE 2

Antibodies against the link peptide of galectin-8 (lp-lec8) or againstrecombinant galectin-8 (rgalectin-8)

Since galectin-8 contains a unique link peptide region, antibodiesagainst this region are not expected to cross-react with othergalectins. A peptide corresponding to positions 168-182 in the linkpeptide of galectin-8 (and an additional Cys residue at the N-terminalsite) of the sequenceCys-Gln-Ile-Ser-Lys-Glu-Thr-Ile-Gln-Lys-Ser-Gly-Lys-Leu-His-Leu (SEQ IDNO:18) was synthesized, purified, and polyclonal antibodies against itwere raised in rabbits by standard procedures. The antibodies (denotedlp-lec8) were affinity-purified over a column of immobilized peptide.lp-lec8 antibodies reacted specifically with galectin-8 both by means ofimmunoprecipitation (IP) and immunoblotting (IB) . Furthermore, theseinteractions could be specifically blocked in the presence of 1 μMpeptide (not shown). Since lp-lec8 antibodies specifically react withthe link peptide of galectin-8, antibodies towards whole recombinantgalectin-8 were generated as well. Purified tag-free rgalectin-8 wasused as immunogen for injection into rabbits, and antibodies wereaffinity purified over columns of Protein A coupled to agarose. Theseantibodies reacted specifically with galectin-8 both by means ofimmunoprecipitation and immunoblotting.

These antibodies are most useful for identification of naturallyoccurring degradation products of galectin-8, where the link peptideregion has been deleted, or proteins homologous to galectin-8 in domainsdifferent from the link peptide region. Cross-reactivity with homologousproteins is assessed by the ability of lp-lec8 antibodies to react withthe suspected candidates, and by the ability of peptides, directedagainst unique regions of galectin-8, outside the link peptide region,to compete with galectin-8 antibody binding.

EXAMPLE 3

In-vitro-translated galectin-8 is biologically active

Galectin-8 cDNA was transcribed and translated in vitro using a TNT(Promega) kit. An ³⁵ S-labeled product of the expected size (34 kDa) wassynthesized (FIG. 4). This in vitro-translated product was indeedgalectin-8 since it could be immunoprecipitated with lp-lec8 antibodiesdescribed in Example 2 (FIG. 4). As predicted by its primary amino acidsequence, in vitro-translated galectin-8 exhibited the key feature ofgalectins, namely, capacity to bind to a column of lactosyl-Sepharose inthe presence of reducing agents, and to be eluted with 0.1 M lactose(not shown).

EXAMPLE 4

Recombinant galectin-8, expressed in bacteria, remains soluble andretains lectin biological activity

To further characterize galectin-8, it was expressed in bacteria as aGST-fusion protein. GST-galectin-8 remained bound toglutathione-Sepharose beads, and could be eluted with glutathione (notshown). GST-galectin-8 retained its sugar-binding capacity and could bepurified by binding to lactosyl-Sepharose and elution with 0.1 M lactose(not shown). Routinely, 3 mg GST-galectin-8 could be purified in such away from 1 liter of bacterial extracts. Like other galectins,GST-galectin-8 also maintained hemagglutination activity. Half andmaximal activities were obtained with 0.1 and 1 μg/ml of GST-galectin-8,respectively.

In a different approach a tag-free rgalectin-8 was expressed employing apET-3a expression plasmid (Novagen) in the pLysS bacterial host. Unlikeintestinal recombinant galectin-4 that precipitates and cannot beextracted with buffers that preserve its lectin activity (12),rgalectin-8 could be readily extracted from bacteria in a soluble formrgalectin-8 was not subjected to major proteolytic cleavage, as itmigrated at the expected size of 34 kDa. Most important, rgalectin-8retained its sugar-binding activity and 1.2 mg protein/liter bacteriawere obtained following its purification over lactosyl-Sepharose column(FIG. 5).

To optimize expression, the induction time and the concentration of IPTGis varied. To further purify GST-galectin-8 or rgalectin-8,approximately 5 mg protein are loaded onto a column of antibodiescovalently linked to Affi-Gel 15 beads (Pharmacia). The bound proteinsare then eluted with HCl/glycine buffer (pH 2.8) and immediatlyneutralized.

EXAMPLE 5

Endogenous galectin-8 is present in rat liver

To demonstrate the presence of endogenous galectin-8 in rat liver, acytosolic (100,000×g supernatant) liver extract was prepared, applied toa column of lactosyl-Sepharose, and proteins retained specifically bythe column were eluted with 0.1 M lactose. Advantage was taken of thefact that hepatic galectin-8 denatures and precipitates upon freezing.Fractions, eluted from the lactosyl-Sepharose column, were thereforefrozen at -20° C., thawed, and centrifuged to precipitate, and thusconcentrate, the hepatic galectin-8. Staining with Coomasie Bluerevealed that most hepatic proteins failed to interact withlactosyl-Sepharose and therefore remained in the flow-through fraction(FIG. 6A). Immunoblotting with lp-lec8 antibodies (FIG. 6B) revealedthat while hepatic galectin-8 could not be detected in total cytosolicliver extracts, a 36 kDa protein, with the expected size of galectin-8,remained bound to, and could be eluted from the lactosyl-Sepharosecolumn. Hepatic galectin-8 was readily detected in the pellets, but notin the supernatants of the (frozen and thawed) eluted fractions,indicating that indeed it denatures upon freezing. These results suggestthat functionally active cytosolic galectin-8 is present in rat liver(FIG. 6).

To estimate the amounts of galectin-8 in rat liver, Triton-soluble liverextracts were prepared, and resolved by means of SDS-PAGE. Known amountsof rgalectin-8 were run in parallel. All samples were then subjected toWestern immunoblotting, using anti-rgalectin-8 antibodies. Assuming thatthe immunoreactivity of rgalectin-8 and the endogenous hepatic proteinare comparable, we calculated that ˜25 ng of galectin-8 are present in100 mg of Triton-soluble liver extracts. These findings suggest thatgalectin-8 comprises ˜0.025% of total Triton-soluble hepatic proteins.

EXAMPLE 6

Galectin-8 is widely expressed. Tissue distribution and cellularlocalization of galectin-8.

Identifying tissues where galectin-8 is highly expressed providesimportant clues related to its possible function and involvement indevelopment. More important, determining whether galectin-8, like othergalectins, is externalized, is of fundamental importance in attempts toassess its mode of action. Three different approaches may be used togain a detailed tissue distribution of galectin-8. i. Northern blotanalysis of rat tissues; ii. to ascertain that the level of mRNA indeedreflects the level of expression of galectin-8, the abundance ofgalectin-8 in various tissues may be determined by Western blot analysisusing anti-rgalectin-8 antibodies. Since galectin-8, like othergalectins, is prone to proteolysis, freshly isolated tissues aredirectly homogenized in 4M guanidinium-HCl to inactivate all proteases.The amount of galectin-8 in the tissue under study is determinedfollowing SDS-PAGE, Western blotting, and probing with anti-rgalectin-8antibodies. iii. In addition, tissues of interest (e. g. liver andbrain) will be studied in more detail by in-situ hybridization. Inpreliminary studies, in situ hybridization of brain slices indicatedthat galectin-8 is specifically expressed in the hypocampus, cerebellum,and brain stem, with little expression in the cortex (not shown). Thesefindings suggest that unlike galectin-4, galectin-8 is an abundantprotein that might play a role in certain brain functions.

Northern blot analysis of rat tissues was carried out and the resultsare shown in Table 1.

                  TABLE I    ______________________________________    Tissue Distribution of galectin-8 mRNA according to    Northern Blot Analysis.    ______________________________________           Lung      100           Liver     43.4           Cardiac muscle                     39.5           Spleen    36.3           Hind limb Muscle                     31.6           Brain     12.6           Fetus     8.1    ______________________________________

Total RNA from the indicated rat tissues was electrophoresed, blotted,and probed as described in legend to FIG. 3. The intensity of the signalcorresponding to the galectin-8 probe was determined by densitometry andis presented as percentage of the strongest signal (normalized to GAPDH)which was obtained in lung (100%).

The expression of galectin-8 in different rat tissues was examined byNorthern blots (FIG. 3). A single mRNA transcript of ˜3 kb hybridizedwith galectin-8 PCR product probe. Unlike galectin-4, which is confinedto intestine and stomach (12), galectin-8 mRNA is highly expressed inlung, and to a lower extent in liver, kidney, spleen, hind-limb, andcardiac muscle (FIG. 3, Table 1). Lower levels of expression weredetected in brain and almost no expression was found in whole ratembryos.

EXAMPLE 7

Generation and purification of recombinant N-terminal (rgalectin-8nt)and C-terminal (rgalectin-8ct) domains of galectin-8.

To determine whether galectin-8nt has any sugar-binding activity, andwhether galectin-8ct might function independently of its N-terminalhalf, galectin-8nt and galectin-8ct are amplified by PCR and properrestriction sites are introduced. Expression of each domain either as aGST-fusion protein or as tag-free domain are carried out as describedabove (Example 4). To express tag-free galectin-8ct the Met residueplaced within the MCS of pET-3d is utilized as the start-site.Purification of galectin-8nt and galectin-8ct is carried out asdescribed above (Example 4).

EXAMPLE 8

Generation of mammalian cells that overexpress galectin-8 in a transientor stable manner.

The cDNA coding for galectin-8 was introduced into four differenteukaryotic high expression plasmids: pcDNA I Amp (Invitrogene); pREP8(Invitrogene); pBPV-II, and pMAMneo (Clontec). The latter plasmid,having a dexamethasone-inducible MMTV-LTR promoter is of particular useif constitutive overexpression of galectin-8 induces growth arrest orprevents adhesion of the transfected cells. Sequencing of thevector/insert boundaries is carried out, to ensure proper integration ofthe insert.

a. Transient expression of galectin-8--Northern blot analysis of RNA andWestern immunoblotting with lp-lec8 antibodies, has indicated that COS-7cells express low levels of endogenous galectin-8. These cells aretherefore appropriate targets to study transient expression ofgalectin-8. COS-7 cells are plated in DMEM/10% FCS at 2×10⁶ cells /10 cmplate, 24 h before transfection. Cells are transfected with 10 μg ofplasmid DNA using DEAE-dextran and DMSO-facilitated uptake according tostandard procedures (modified by 0.1 mM chloriquine treatment). Cellsare harvested 48-72 h thereafter, and the expressed galectin-8 isdetected by Western immunoblotting with lp-lec8 or rgalectin-8antibodies. Galectin-8 is purified by affinity-chromatography overlactosyl-Sepharose column, and by immunoaffinity chromatography usinglp-lec8 or rgalectin-8 antibodies coupled to Sepharose asimmunoadsorbent.

b. Stable expression of galectin-8--The above expression plasmids areused for stable transfection of galectin-8 DNA into Chinese HamsterOvary (CHO) cells that have relatively low amount of endogenousgalectin-8. Stable transfectants are identified by their ability toaccumulate galectin-8 in the cytosol, or to secrete galectin-8 into themedium. Conditioned-medium is collected, concentrated by AmiconCentricon-10 micro concentrator, and lyophilized. Cytosolic extracts areprepared by boiling in "sample buffer" and the presence of galectin-8 isdetected by immunoblotting with galectin-8 antibodies. Cells expressingthe highest concentration of galectin-8 are further propagated.

EXAMPLE 9

Biological activity of whole rgalectin-8 and its individually-expressedN- or C-terminal domains

To assess the functional need for two CRDs within the same polypeptidechain of galectin-8, the biological activity of rgalectin-8 is comparedwith that of its individually-expressed domains.

i. Hemagglutination activity of rgalectin-8, rgalectin-8nt andrgalectin-8ct is assayed as previously described (17). Rabbiterythrocytes are trypsin-treated and fixed with glutaraldehyde.Following washings in 0.1 M glycine/PBS and PBS, and proper dilution,hemagglutination activity of serial dilutions of rgalectin-8 is comparedwith those of rgalectin-8nt and rgalectin-8ct. If rgalectin-8, likegalectin-1, is capable of forming homodimers, and if both CRDs ofgalectin-8 are capable of sugar binding, then rgalectin-8 is expected toexpress hemagglutination activity. If however rgalectin-8nt has reducedor no sugar-binding activity, and if rgalectin-8 fails to dimerize, thenrgalectin-8, having a single functional CRD at the C-terminal domain,might fail to express hemagglutination activity. These results willimplicate galectin-8 as having a function different from cross-linkingglycoconjugates.

ii. Carbohydrate-binding specificity of whole galectin-8 and itsindividually-expressed domains is compared to previously determinedspecificity of other galectins, including galectin-4. To avoid possiblealterations in the native structure of galectin-8 (e.g. due tocarboxymethylation and iodination) 5 μg of purified rgalectin-8 (orindividual domains) are incubated with 100 μl of lactosyl-Sepharose;conditions that result in quantitative binding of rgalectin-8. Bindingspecificity may be determined by the capacity of various saccharides(e.g. thiodigalactose, thiodiglucose) to inhibit binding of rgalectin-8(or individual domains), when compared with lactose. If galectin8ntexpresses, as predicted, altered or markedly reducedcarbohydrate-binding activity, binding activity may be restored bysite-directed mutagenesis, where the Ile-90 residue is mutated to Arg.

EXAMPLE 10

Site-directed mutagenesis.

Site-directed mutagenesis is carried out using "Altered Sites II invitro mutagenesis systems" (Promega) according to the manufacturer'smanual. First, Ile-90 is mutated to Arg to determine how suchsubstitution affects hemagglutination activity and sugar bindingspecificity of rgalectin-8nt and whole galectin-8. Conversely, Arg-253,located within the WG-E-R motif at the C-terminal CRD may be mutated toIle, and the effect of this mutation on the biological activity ofgalectin-8 is assessed. If Arg-253→Ile mutation markedly reduces orabolishes the in vitro biological activity of galectin-8, then thebiological consequences of overexpression of this negative-dominantmutant will be compared with cells that overexpress the native form ofgalectin-8.

EXAMPLE 11

Sensitivity of rgalectin-8 to oxidation.

One whole mark of certain galectins is the sensitivity of theircarbohydrate-binding activity to oxidation. Other studies suggest thatfor certain of these lectins the thiol-dependence may be ascribed to anartifact of the extraction procedure rather than an intrinsicrequirement of the protein itself. To assess whether galectin-8 requiresreducing environment to remain biologically active, the effects ofvarious reductants and oxidants on the binding activity of galectin-8 tolactosyl-Sepharose are studied as described for other galectins. Ifgalectin-8 activity is sensitive to oxidation, alkylation of rgalectin-8may be carried out with iodoacetamide or with N-ethyl-maleimide. Themodified product is then subjected to rechromatography overlactosyl-Sepharose column and is eluted with water. Alkylation, thatstabilizes galectin-1, may preserve and stabilize rgalectin-8 activity(i.e. binding affinity to lactosyl-Sepharose), and enables increase ofthe half-life of rgalectin-8 and better study of its effects on culturedcells under the oxidizing environment of tissue culture medium.

EXAMPLE 12

Sensitivity of rgalectin-8 to proteolysis.

Preliminary experiments have indicated that endogenous mammaliangalectin-8 is susceptible to proteolysis. To determine the physiologicalsignificance of this phenomena, pulse-chase experiments with ³⁵S-labeled cells, followed by immunoprecipitation of the endogenousgalectin-8, are carried out in CHO cells overexpressing galectin-8. ³⁵S-labeled galectin-8 is precipitated with lp-lec8 or rgalectin-8antibodies. The half-life of endogenous galectin-8 and the formation ofin vivo degradation products are then evaluated. To distinguishproteolysis that occurs in vivo from one that occurs during extractionand purification, homogenization is carried out in the presence of traceamounts of ¹²⁵ I-labeled rgalectin-8.

EXAMPLE 13

Biological activity of galectin-8

The effects of galectin-8 on cell adhesion and on regulation of cellulargrowth are examined.

Effects of galectin-8 on cell adhesion

One of the well characterized effects of galectin-1 is its ability toinhibit myoblast adhesion to laminin (15). To determine whethergalectin-8 shares a similar property, the effects of overexpression ofgalectin-8 on cell adhesion are studied. COS-7 cells are co-transfectedwith an expression vector for β-galactosidase (pSMβGal) at a 1:20 ratioto the galectin-8 vector. Cells expressing β-galactosidase are easilydistinguished by a blue staining after histochemical reaction withX-gal, 36 h following transfection. Alterations in adhesion of bluecells as a function of time are monitored. Control cells arecotransfected with pSMβGal and pcDNA-IR (which contains an insertencoding for the insulin receptor). If positive results are obtained,thio-D-glucose (TDG) is added to inhibit lectin-carbohydrateinteractions and study the contribution of the carbohydrate-bindingdomains to this effect.

In an alternative approach CHO cells, transfected with thepMAMneo-galectin-8 plasmid (which has a dexamethasone-inducible MMTV-LTRpromoter) is used. Their adhesive properties to the culture dish, beforeand after induction, are compared. If positive results are obtained, theeffects of TDG on cell adhesion and the effects of exogenously-addedrgalectin-8 on non-induced cells are determined.

Function of galectin-8 as a cytostatic factor and cell growth regulator.

mGBP, a single-domain homologue of galectin-8, was shown to be a cellgrowth-regulatory molecule and a cytostatic factor that binds to aspecific cell surface receptor (8). To determine whether galectin-8fulfills a similar role, since galectin-8 is expressed in rat liver, rathepatoma (Fao) cells are used as a model system. Another model is mouseembryo fibroblasts (MEF), that were already shown to be subjected to thegrowth inhibitory action of mGBP (8). Growth inhibition induced bypurified rgalectin-8 is assessed by several parameters:

i. Direct counting of logarithmically growing cells, incubated forincreasing time periods with increasing concentrations of native ordenatured (control) rgalectin-8. Cell viability is assessedcalorimetrically utilizing the neutral red uptake assay.

ii. Inhibition of DNA synthesis is monitored by ³ H! thymidineincorporation into control, and rgalectin-8-treated cells.

iii. Change in population distribution, due to inhibition of cellgrowth, is assessed by FACS analysis.

iv. Changes in cell morphology are monitored in cells grown on coverslips. Following treatment, cells are washed, fixed, and viewed byNomarski interference contrast microscopy.

The reversibility of the galectin-8 effects on these parameters may thenbe evaluated. The relation between sugar binding and the biologicalactivity of rgalectin-8 may be further assessed by the ability of 10 mMTDG to compete for rgalectin-8 binding. Successful results lead to thesecond stage of the study, where it is determined whether growthinhibition is related to the growth state, as is in the case of mGBP andcytokines. For that purpose cells stationed in Go by serum starvation,and cells rescued from Go by serum stimulation, are treated withgalectin-8 for different times, and its potency to attenuate or inhibitcell growth is evaluated.

Inhibition of DNA synthesis was monitored in control andrgalectin-8-treated CHO cells as described in the legend to FIG. 7. Itcan be seen that rgalectin-8 inhibits serum-induced ³ H! thymidineincorporation in a dose-dependent manner. Half-maximal effects areobtained at 0.5 μM and maximal effects at 2 μM rgalectin-8, GST alone iswithout effect.

EXAMPLE 14

Use of galectin-8 antibodies as diagnostic tools for neoplastictransformation.

Suitable compositions prepared by well-known standard procedures,containing anti-galectin-8 antibodies may be used to detectoverexpression of this protein following neoplastic transformation ingeneral, and in metastatic melanoma cells in particular, andaccordingly, to determine whether overexpression of galectin-8 can serveas an early signal for neoplastic transformation, and/or the developmentof metastatic melanoma. Thus, the anti-galectin-8 antibodies mat serveas a diagnostic tool for early detection of the above disease. Moreover,the presence of a subject's own anti-galectin-8 antibodies can alsoserve as such a diagnostic tool, which endogenous anti-galectin-8antibodies may be assayed with purified galectin-8.

REFERENCES

1. Sharon, N. (1993) TIBS 18, 221-225.

2. Barondes, S. H., Castronovo, V., Cooper, D. N., Cummings, R. D.,Drickamer, K., Feizi, T., Gitt, M. A., Hirabayashi, J., Hughes, C.,Kasai, K. et. al. (1994) Cell 76, 597-598.

3. Barondes, S. H., Cooper, D. N. W., Gitt, M. A., and Leffler, H.(1994) J. Biol. Chem. 269, 20807-20810.

4. Hirabayashi, J. and Kasai, K. (1991) J Biol Chem 266, 23648-23653.

5. Lobsanov, Y. D., Gitt, M. A., Leffler, H., Barondes, S. H., and Rini,J. M. (1993) J Biol Chem 268, 27034-27038.

6. Tracey, B. M., Feizi, T., Abbott, W. M., Carruthers, R. A., Green, B.N. and Lawson, A. M. (1992) J Biol Chem 267, 10342-10347.

7. Cooper, D. N., Massa, S. M. and Barondes, S. H. (1991) J Cell Biol115, 1437-1448.

8. Wells, V., and Mallucci, L. (1991) Cell 64, 91-97.

9. Yamaoka, K., Ohno, S., Kawasaki, H. and Suzuki, K. (1991) BiochemBiophys Res Commun 179, 272-279.

10. Poirier, F., Timmons, P. M., Chan, C. T., Guenet, J. L. and Rigby,P. W. (1992) Development 115, 143-155.

11. Ochieng, J., Platt, D., Tait, L., Hogan, V., Raz, T., Carmi, P., andRaz, A. (1993) Biochemistry 32, 4455-4460.

12. Oda, Y., Herrmann, J., Gitt, M. A., Turck, C. W., Burlingame, A. L.,Barondes, S. H. and Leffler, H. (1993) J Biol Chem 268, 5929-5939.

13. Hirabayashi, J., Satoh, M. and Kasai, K. (1992) J Biol Chem 267,15485-15490.

14. Lamphere, L. and Lienhard, G. E. (1992) Endocrinology 131,2196-2202.

15. Cooper, D. N., and Barondes, S. H. (1990) J Cell Biol 110,1681-1691.

16. Sambrook, J., Fritsch, E. F., and Maniatis, T. Molecular Cloning, alaboratory manual (Cold Spring Harbor Laboratory Press, 1989).

17. Lis, H., and Sharon, N. (1972) Methods Enzymol. 28, 360-368.

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#u Leu Tyr Ala His Arg    #       125    - ATC AAC CCA GAG AAG ATA GAC ACA CTG GGC AT - #C TTC GGC AAA GTG AAC     552    Ile Asn Pro Glu Lys Ile Asp Thr Leu Gly Il - #e Phe Gly Lys Val Asn    #   140    - ATT CAC TCC ATC GGG TTC AGA TTC AGC TCG GA - #T TTA CAG AGT ATG GAA     600    Ile His Ser Ile Gly Phe Arg Phe Ser Ser As - #p Leu Gln Ser Met Glu    145                 1 - #50                 1 - #55                 1 -    #60    - ACA TCT ACT CTG GGA CTG ACA CAG ATA AGT AA - #A GAA AAT ATA CAA AAG     648    Thr Ser Thr Leu Gly Leu Thr Gln Ile Ser Ly - #s Glu Asn Ile Gln Lys    #               175    - TCT GGC AAG CTC CAT TTG AGC CTG CCA TTT GA - #A GCA AGG TTG AAT GCC     696    Ser Gly Lys Leu His Leu Ser Leu Pro Phe Gl - #u Ala Arg Leu Asn Ala    #           190    - TCC ATG GGC CCT GGA CGA ACC GTT GTC GTT AA - #A GGA GAA GTG AAT ACA     744    Ser Met Gly Pro Gly Arg Thr Val Val Val Ly - #s Gly Glu Val Asn Thr    #       205    - AAT GCC ACA AGC TTT AAT GTT GAC CTA GTG GC - #A GGA AGG TCA AGG GAT     792    Asn Ala Thr Ser Phe Asn Val Asp Leu Val Al - #a Gly Arg Ser Arg Asp    #   220    - ATC GCT CTG CAC TTG AAC CCA CGC CTG AAT GT - #G AAA GCG TTT GTA AGA     840    Ile Ala Leu His Leu Asn Pro Arg Leu Asn Va - #l Lys Ala Phe Val Arg    225                 2 - #30                 2 - #35                 2 -    #40    - AAC TCC TTT CTT CAG GAT GCC TGG GGA GAA GA - #G GAG AGA AAC ATT ACC     888    Asn Ser Phe Leu Gln Asp Ala Trp Gly Glu Gl - #u Glu Arg Asn Ile Thr    #               255    - TGC TTC CCA TTT AGT TCT GGG ATG TAC TTT GA - #G ATG ATA ATT TAC TGT     936    Cys Phe Pro Phe Ser Ser Gly Met Tyr Phe Gl - #u Met Ile Ile Tyr Cys    #           270    - GAT GTC CGA GAG TTC AAG GTT GCA GTA AAT GG - #T GTG CAC AGC CTG GAG     984    Asp Val Arg Glu Phe Lys Val Ala Val Asn Gl - #y Val His Ser Leu Glu    #       285    - TAC AAG CAC AGA TTT AAA GAC CTA AGC AGC AT - #C GAC ACA CTA GCA GTT    1032    Tyr Lys His Arg Phe Lys Asp Leu Ser Ser Il - #e Asp Thr Leu Ala Val    #   300    - GAT GGC GAT ATC CGT TTG CTG GAT GTA AGG AG - #C TGG TAGCTATCAT    1078    Asp Gly Asp Ile Arg Leu Leu Asp Val Arg Se - #r Trp    305                 3 - #10                 3 - #15    - GACTGCCAGA ACCCTGGAAA TACAAAATGG CTTATCCGAT ACTGGCCATG TC - #AAATGCAT    1138    - CTCGCTTTCA CCACATTGTT ATACTGTTAA GTTGAGCTCG CACAACATCA AG - #TCCTACTG    1198    #             1247GCCAT GCAGTGTGGC TACCTCTGAA TTCCCAGGA    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 316 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    - Met Leu Ser Leu Ser Asn Leu Gln Asn Ile Il - #e Tyr Asn Pro Thr Ile    #                 15    - Pro Tyr Val Ser Thr Ile Thr Glu Gln Leu Ly - #s Pro Gly Ser Leu Ile    #             30    - Val Ile Arg Gly His Val Pro Lys Asp Ser Gl - #u Arg Phe Gln Val Asp    #         45    - Phe Gln His Gly Asn Ser Leu Lys Pro Arg Al - #a Asp Val Ala Phe His    #     60    - Phe Asn Pro Arg Phe Lys Arg Ser Asn Cys Il - #e Val Cys Asn Thr Leu    # 80    - Thr Asn Glu Lys Trp Gly Trp Glu Glu Ile Th - #r His Asp Met Pro Phe    #                 95    - Arg Lys Glu Lys Ser Phe Glu Ile Val Ile Me - #t Val Leu Lys Asn Lys    #           110    - Phe His Val Ala Val Asn Gly Lys His Ile Le - #u Leu Tyr Ala His Arg    #       125    - Ile Asn Pro Glu Lys Ile Asp Thr Leu Gly Il - #e Phe Gly Lys Val Asn    #   140    - Ile His Ser Ile Gly Phe Arg Phe Ser Ser As - #p Leu Gln Ser Met Glu    145                 1 - #50                 1 - #55                 1 -    #60    - Thr Ser Thr Leu Gly Leu Thr Gln Ile Ser Ly - #s Glu Asn Ile Gln Lys    #               175    - Ser Gly Lys Leu His Leu Ser Leu Pro Phe Gl - #u Ala Arg Leu Asn Ala    #           190    - Ser Met Gly Pro Gly Arg Thr Val Val Val Ly - #s Gly Glu Val Asn Thr    #       205    - Asn Ala Thr Ser Phe Asn Val Asp Leu Val Al - #a Gly Arg Ser Arg Asp    #   220    - Ile Ala Leu His Leu Asn Pro Arg Leu Asn Va - #l Lys Ala Phe Val Arg    225                 2 - #30                 2 - #35                 2 -    #40    - Asn Ser Phe Leu Gln Asp Ala Trp Gly Glu Gl - #u Glu Arg Asn Ile Thr    #               255    - Cys Phe Pro Phe Ser Ser Gly Met Tyr Phe Gl - #u Met Ile Ile Tyr Cys    #           270    - Asp Val Arg Glu Phe Lys Val Ala Val Asn Gl - #y Val His Ser Leu Glu    #       285    - Tyr Lys His Arg Phe Lys Asp Leu Ser Ser Il - #e Asp Thr Leu Ala Val    #   300    - Asp Gly Asp Ile Arg Leu Leu Asp Val Arg Se - #r Trp    305                 3 - #10                 3 - #15    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 135 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    - Met Ala Gly Gly Leu Val Ala Ser Asn Leu As - #n Leu Lys Pro Gly Glu    #                15    - Cys Leu Arg Val Arg Gly Glu Val Ala Pro As - #p Ala Lys Ser Glu Val    #            30    - Leu Asn Leu Gly Lys Asp Ser Asn Asn Leu Cy - #s Glu His Glu Asn Pro    #        45    - Arg Glu Asn Ala His Gly Asp Ala Asn Thr Il - #e Val Cys Asn Ser Lys    #    60    - Asp Gly Gly Ala Trp Gly Thr Glu Gln Arg Gl - #u Ala Val Phe Pro Glu    #80    - Gln Pro Gly Ser Val Ala Glu Val Cys Ile Th - #r Phe Asp Gln Ala Asn    #                95    - Glu Thr Val Lys Leu Pro Asp Gly Tyr Glu Ph - #e Lys Ser Pro Asn Arg    #           110    - Leu Asn Leu Glu Ala Ile Asn Tyr Met Ala Al - #a Asp Gly Asp Phe Lys    #       125    - Ile Lys Cys Val Ala Phe Asp    #   135    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 132 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    - Met Thr Gly Glu Leu Glu Val Lys Asn Met As - #p Met Lys Pro Gly Ser    #                15    - Thr Leu Lys Ile Thr Gly Ser Ile Ala Asp Gl - #y Thr Asp Gly Glu Val    #            30    - Ile Asn Leu Gly Gln Gly Thr Asp Lys Leu As - #n Glu His Glu Asn Pro    #        45    - Arg Glu Ser Glu Ser Thr Ile Val Cys Asn Se - #r Leu Asp Gly Ser Asn    #    60    - Trp Gly Gln Glu Gln Arg Glu Asp His Leu Cy - #s Glu Ser Pro Gly Ser    #80    - Glu Val Lys Phe Thr Val Thr Phe Glu Ser As - #p Lys Glu Lys Val Lys    #                95    - Leu Pro Asp Gly His Glu Leu Thr Ser Pro As - #n Arg Leu Gly His Ser    #           110    - His Leu Ser Tyr Leu Ser Trp Arg Gly Gly Ph - #e Asn Pro Ser Ser Phe    #       125    - Lys Leu Lys Glu        130    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 135 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    - Val Pro Tyr Asp Met Pro Leu Pro Gly Gly Va - #l Met Pro Arg Met Leu    #                15    - Ile Thr Ile Ile Gly Thr Val Lys Pro Asn Al - #a Asn Ser Glu Thr Leu    #            30    - Asn Glu Lys Lys Gly Asn Asp Ile Ala Glu Hi - #s Glu Asn Pro Arg Glu    #        45    - Asn Glu Asn Asn Arg Arg Val Ile Val Cys As - #n Thr Lys Gln Asp Asn    #    60    - Asn Trp Gly Arg Glu Glu Arg Gln Ser Ala Ph - #e Pro Glu Glu Ser Gly    #80    - Lys Pro Glu Lys Ile Gln Val Leu Val Glu Al - #a Asp His Glu Lys Val    #                95    - Ala Val Asn Asp Val His Leu Leu Gln Tyr As - #n His Arg Met Lys Asn    #           110    - Leu Arg Glu Ile Ser Gln Leu Gly Ile Ile Gl - #y Asp Ile Thr Leu Thr    #       125    - Ser Ala Ser His Ala Met Ile    #   135    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 177 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    - Met Ala Tyr Val Pro Ala Pro Gly Tyr Gln Pr - #o Thr Tyr Asn Pro Thr    #                15    - Leu Pro Tyr Lys Arg Pro Ile Pro Gly Gly Le - #u Ser Val Gly Met Ser    #            30    - Ile Tyr Ile Gln Gly Ile Ala Lys Asp Asn Me - #t Arg Arg Glu His Val    #        45    - Asn Glu Ala Val Gly Gln Asp Glu Gly Ala As - #p Ile Ala Glu His Glu    #    60    - Asn Pro Arg Glu Asp Gly Trp Asp Lys Val Va - #l Phe Asn Thr Met Gln    #80    - Ser Gly Gln Trp Gly Lys Glu Glu Lys Lys Ly - #s Ser Met Pro Glu Gln    #                95    - Lys Gly His His Glu Glu Leu Val Glu Met Va - #l Met Ser Glu His Lys    #           110    - Lys Val Val Val Asn Gly Thr Pro Phe Tyr Gl - #u Tyr Gly His Arg Leu    #       125    - Pro Leu Gln Met Val Thr His Leu Gln Val As - #p Gly Asp Leu Glu Leu    #   140    - Gln Ser Ile Asn Phe Leu Gly Gly Gln Pro Al - #a Ala Ser Gln Tyr Pro    145                 1 - #50                 1 - #55                 1 -    #60    - Gly Thr Met Thr Ile Pro Ala Tyr Pro Ser Al - #a Gly Tyr Asn Pro Pro    #               175    - Gln    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 147 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    - Met Asn Ser Leu Pro Val Met Ala Gly Pro Pr - #o Ile Phe Asn Pro Pro    #                15    - Val Pro Tyr Val Gly Thr Leu Gln Gly Gly Le - #u Thr Ala Arg Arg Thr    #            30    - Ile Ile Ile Lys Gly Tyr Val Leu Pro Thr Al - #a Lys Asn Ile Ile Ile    #        45    - Asn Glu Lys Val Gly Ser Thr Gly Asp Ile Al - #a Glu His Met Asn Pro    #    60    - Arg Ile Gly Asp Cys Val Val Arg Asn Ser Ty - #r Met Asn Gly Ser Trp    #80    - Gly Ser Glu Glu Arg Lys Ile Pro Tyr Asn Pr - #o Glu Gly Ala Gly Gln    #                95    - Phe Glu Asp Leu Ser Ile Arg Cys Gly Thr As - #p Arg Glu Lys Val Phe    #           110    - Ala Asn Gly Gln His Leu Phe Asp Arg Ser Hi - #s Arg Phe Gln Ala Pro    #       125    - Gln Arg Val Asp Met Leu Glu Ile Lys Gly As - #p Ile Thr Leu Ser Tyr    #   140    - Val Gln Ile    145    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 146 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    - Met Ser Ala Glu Glu Pro Lys Ser Tyr Pro Va - #l Pro Tyr Arg Ser Val    #                15    - Leu Gln Glu Lys Leu Glu Pro Gly Gln Thr Le - #u Ile Val Lys Gly Ser    #            30    - Thr Ile Asp Glu Ser Gln Arg Glu Thr Ile As - #n Leu His Ser Lys Thr    #        45    - Ala Asp Phe Ser Gly Asn Asp Val Pro Leu Hi - #s Val Ser Val Arg Glu    #    60    - Asp Glu Gly Lys Ile Val Leu Asn Ser Phe Se - #r Asn Gly Glu Trp Gly    #80    - Lys Glu Glu Arg Lys Ser Asn Pro Ile Lys Ly - #s Gly Asp Ser Glu Asp    #                95    - Ile Arg Ile Arg Ala His Asp Asp Arg Glu Gl - #n Ser Ile Val Asp His    #           110    - Lys Glu Phe Lys Asp Tyr Glu His Arg Leu Pr - #o Leu Ser Ser Ile Ser    #       125    - His Leu Ser Ile Asp Gly Asp Leu Tyr Leu As - #n His Val His Trp Gly    #   140    - Gly Lys    145    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 131 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    - Pro Val Pro Tyr Glu Ser Gly Leu Ala Asn Gl - #y Leu Pro Val Gly Lys    #                15    - Ser Leu Leu Val Phe Gly Thr Val Glu Lys Ly - #s Ala Lys Arg Glu His    #            30    - Val Asn Leu Leu Arg Lys Asn Gly Asp Ile Se - #r Glu His Glu Asn Pro    #        45    - Arg Glu Asp Glu Lys His Val Val Arg Asn Se - #r Leu Ala Ala Asn Glu    #    60    - Trp Gly Asn Glu Glu Arg Glu Gly Lys Asn Pr - #o Glu Glu Lys Gly Val    #80    - Gly Glu Asp Leu Val Ile Gln Asn Glu Glu Ty - #r Ala Glu Gln Val Phe    #                95    - Val Asn Gly Glu Arg Tyr Ile Ser Arg Ala Hi - #s Arg Ala Asp Pro His    #           110    - Asp Ile Ala Gly Leu Gln Ile Ser Gly Asp Il - #e Glu Leu Ser Gly Ile    #       125    - Gln Ile Gln        130    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 184 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    - Met Leu Ser Leu Ser Asn Leu Gln Asn Ile Il - #e Tyr Asn Pro Thr Ile    #                15    - Pro Tyr Val Ser Thr Leu Thr Glu Gln Leu Ly - #s Pro Gly Ser Leu Ile    #            30    - Val Ile Arg Gly His Val Pro Lys Asp Ser Gl - #u Arg Glu Gln Val Asp    #        45    - Glu Gln His Gly Asn Ser Leu Lys Pro Arg Al - #a Asp Val Ala Glu His    #    60    - Glu Asn Pro Arg Glu Lys Arg Ser Asn Cys Il - #e Val Cys Asn Thr Leu    #80    - Thr Asn Glu Lys Trp Gly Trp Glu Glu Ile Th - #r His Asp Met Pro Glu    #                95    - Arg Lys Glu Lys Glu Glu Glu Ile Val Ile Me - #t Val Leu Lys Asn Lys    #           110    - Glu His Val Ala Val Asn Gly Lys His Ile Le - #u Leu Tyr Ala His Arg    #       125    - Ile Asn Pro Glu Lys Ile Asp Thr Leu Gly Il - #e Phe Gly Lys Val Asn    #   140    - Ile His Ser Ile Gly Phe Arg Phe Ser Ser As - #p Leu Gln Ser Met Glu    145                 1 - #50                 1 - #55                 1 -    #60    - Thr Ser Thr Leu Gly Leu Thr Gln Ile Ser Ly - #s Glu Asn Ile Gln Lys    #               175    - Ser Gly Lys Leu His Leu Ser Leu                180    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 132 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    - Pro Glu Glu Ala Arg Leu Asn Ala Ser Met Gl - #y Pro Gly Arg Thr Val    #                15    - Val Val Lys Gly Glu Val Asn Thr Asn Ala Th - #r Ser Glu Asn Val Asp    #            30    - Leu Val Ala Gly Arg Ser Arg Asp Ile Ala Il - #e His Ile Asn Pro Arg    #        45    - Ile Asn Val Lys Ala Phe Val Arg Asn Ser Ph - #e Leu Gln Asp Ala Trp    #    60    - Gly Glu Glu Glu Arg Asn Ile Thr Cys Phe Pr - #o Glu Ser Ser Gly Met    #80    - Tyr Glu Glu Met Ile Ile Tyr Cys Asp Val Ar - #g Glu Glu Lys Val Ala    #                95    - Val Asn Gly Val His Ser Leu Glu Tyr Lys Hi - #s Arg Phe Lys Asp Leu    #           110    - Ser Ser Ile Asp Thr Leu Ala Val Asp Gly As - #p Ile Arg Leu Leu Asp    #       125    - Val Arg Ser Trp        130    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 15 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    - Cys Tyr Ala Ser Ile Asn Phe Gln Lys Gln Pr - #o Glu Asp Arg Gln    #                15    - (2) INFORMATION FOR SEQ ID NO:13:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 25 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    #               25 GTCA GTACC    - (2) INFORMATION FOR SEQ ID NO:14:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 21 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    #21                CAAC A    - (2) INFORMATION FOR SEQ ID NO:15:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 30 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    #           30     TGTC CTTAAGCAAT    - (2) INFORMATION FOR SEQ ID NO:16:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 27 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    #             27   CCTT AAGCAAT    - (2) INFORMATION FOR SEQ ID NO:17:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 30 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    #           30     TTTT GTATTTCCAG    - (2) INFORMATION FOR SEQ ID NO:18:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 16 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    - Cys Gln Ile Ser Lys Glu Thr Ile Gln Lys Se - #r Gly Lys Leu His Leu    #                15    - (2) INFORMATION FOR SEQ ID NO:19:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 6 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    - His Phe Asn Pro Arg Leu    1               5    __________________________________________________________________________

I claim:
 1. A sugar-binding and cell agglutinating molecule selectedfrom the group consisting of:(i) the protein galectin-8 having the aminoacid sequence of SEQ ID NO:2; (ii) a protein other than the protein of(i), extracted from a mammal of a species other than rat and which hassugar-binding ability and the ability to agglutinate formaldehyde-fixed,trypsin-treated rabbit erythrocyte, which protein is encoded by DNAwhich hybridizes to the DNA of SEQ ID NO:1 under moderately stringentconditions carried out at 42° C. in 50% formamide 5×SSC with washes at60° C. in 0.1×SSC, 0.1% SDS; (iii) a protein other than the protein of(i), extracted from a mammal of a species other than rat and which hassugar-binding ability and the ability to agglutinate formaldehyde-fixed,trypsin-treated rabbit erythrocyte, which protein is bound by anantibody specific for an epitope in the region of amino acids 153-184 ofSEQ ID NO:2; and (iv) a fragment of (i), (ii) or (iii) which hassugar-binding ability and the ability to agglutinate formaldehyde-fixed,trypsin-treated rabbit erythrocyte.
 2. A molecule in accordance withclaim 1, comprising the galectin-8 protein of (i).
 3. An isolatedrecombinant DNA molecule comprising a nucleotide sequence encoding amolecule in accordance with claim
 1. 4. An isolated DNA molecule inaccordance with claim 3, comprising the nucleotide sequence ofnucleotides 121-1068 of SEQ ID NO:1.
 5. An isolated DNA molecule inaccordance with claim 3, comprising the nucleotide sequence of thecoding region of the galectin-8 gene.
 6. A recombinant expression vectorcomprising a recombinant DNA molecule in accordance with claim
 3. 7. Ahost cell containing a recombinant expression vector in accordance withclaim
 6. 8. A process for producing a sugar-binding molecule, comprisingculturing a host cell according to claim 7 under conditions promotingexpression, and isolating the sugar-binding molecule expressed thereby.9. An antibody specific for an epitope in the region of 153-184 of SEQID NO:2.